Increased Resistance to Biotrophic Pathogens in the
Arabidopsis Constitutive Induced Resistance 1 Mutant Is
EDS1 and PAD4-Dependent and Modulated by
Environmental Temperature
Maryke Carstens1¤, Tyronne K. McCrindle1, Nicolette Adams1, Anastashia Diener1, Delroy T. Guzha1,
Shane L. Murray1, Jane E. Parker2, Katherine J. Denby3, Robert A. Ingle1*
1 Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, South Africa, 2 Department of Plant Microbe Interactions, Max Planck Institute for
Plant Breeding Research, Köln, Germany, 3 School of Life Sciences and Warwick Systems Biology Centre, University of Warwick, Coventry, United Kingdom
Abstract
The Arabidopsis constitutive induced resistance 1 (cir1) mutant displays salicylic acid (SA)-dependent constitutive expression
of defence genes and enhanced resistance to biotrophic pathogens. To further characterise the role of CIR1 in plant
immunity we conducted epistasis analyses with two key components of the SA-signalling branch of the defence network,
ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and PHYTOALEXIN DEFICIENT4 (PAD4). We demonstrate that the constitutive
defence phenotypes of cir1 require both EDS1 and PAD4, indicating that CIR1 lies upstream of the EDS1-PAD4 regulatory
node in the immune signalling network. In light of this finding we examined EDS1 expression in cir1 and observed increased
protein, but not mRNA levels in this mutant, suggesting that CIR1 might act as a negative regulator of EDS1 via a posttranscriptional mechanism. Finally, as environmental temperature is known to influence the outcome of plant-pathogen
interactions, we analysed cir1 plants grown at 18, 22 or 25uC. We found that susceptibility to Pseudomonas syringae pv.
tomato (Pst) DC3000 is modulated by temperature in cir1. Greatest resistance to this pathogen (relative to PR-1:LUC control
plants) was observed at 18uC, while at 25uC no difference in susceptibility between cir1 and control plants was apparent.
The increase in resistance to Pst DC3000 at 18uC correlated with a stunted growth phenotype, suggesting that activation of
defence responses may be enhanced at lower temperatures in the cir1 mutant.
Citation: Carstens M, McCrindle TK, Adams N, Diener A, Guzha DT, et al. (2014) Increased Resistance to Biotrophic Pathogens in the Arabidopsis Constitutive
Induced Resistance 1 Mutant Is EDS1 and PAD4-Dependent and Modulated by Environmental Temperature. PLoS ONE 9(10): e109853. doi:10.1371/journal.pone.
0109853
Editor: Boris A. Vinatzer, Virginia Tech, United States of America
Received July 23, 2014; Accepted September 4, 2014; Published October 10, 2014
Copyright: ß 2014 Carstens et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its
Supporting Information files.
Funding: This research was funded by a grant (IFR2011032900062) from the National Research Foundation, South Africa (www.nrf.ac.za) and by the University of
Cape Town (www.uct.ac.za). Experiments carried out in Jane Parker’s lab were funded by the Max-Planck Society (www.mpg.de) and an Alexander von Humboldt
Foundation (www.humboldt-foundation.de/web/home.html) ‘Sofja Kovaleskaja’ award. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* Email: [email protected]
¤ Current address: Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria, South Africa
response [1,3]. The final layer of innate immunity is systemic
acquired resistance (SAR), whereby infection of one part of a plant
leads to increased resistance of uninfected tissues to subsequent
pathogen challenge [4]. SAR is thought to be established by coordinated expression of an array of anti-microbial pathogenesis
related (PR) genes [4,5]. All three branches of innate immunity
rely on large scale transcriptional re-programming of the host
plant, activated via a complex network that is influenced by the
crosstalk between salicylic acid (SA), jasmonic acid (JA) and
ethylene (Et) signalling [6,7].
Two key signalling components in the SA-signalling branch of
the defence network are ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and PHYTOALEXIN DEFICIENT4 (PAD4)
[8,9], both of which show homology to eukaryotic acyl lipases. The
EDS1-PAD4 node has long been recognised as a central regulator
Introduction
Plants have a robust innate immune system that affords
protection against attack by potential pathogens in their local
environment. Detection of pathogen associated molecular patterns
(PAMPs) such as flagellin by pattern recognition receptors at the
plasma membrane leads to activation of PAMP-triggered immunity (PTI) [1,2]. Successful phytopathogens have evolved mechanisms, including effectors, to subvert or suppress PTI, allowing
them to successfully colonise the plant host [1,3]. This in turn led
to the evolution of effector-triggered immunity (ETI) in plants,
which relies on the direct or indirect detection of pathogen
effectors by cognate host resistance (R) proteins [1,4]. While there
is a significant overlap between these two branches of the innate
immune system, ETI is generally regarded as a stronger and more
rapid response, and is associated with the hypersensitive (HR)
PLOS ONE | www.plosone.org
1
October 2014 | Volume 9 | Issue 10 | e109853
CIR1-Mediated Immunity in Arabidopsis Requires EDS1 and PAD4
constitutive disease resistance are the constitutive expressor of PR
genes (cpr) mutants [20–22]. These mutants display SA signallingdependent constitutive expression of PR genes and enhanced
resistance to virulent biotrophic pathogens [21]. The cpr-type
mutants can broadly be divided into two groups [23], those that
display constitutive HR-like cell death such as cpr5 and lsd1, and
those that do not, including cpr1 and dnd1. The cir1 (constitutively
induced resistance 1) mutant belongs to the second class of cpr
mutants, and was identified in a mutant screen for increased
luciferase activity in Col-0 plants carrying a PR-1:LUC reporter
[24]. The cir1 mutation is recessive, and homozygous cir1 plants
display increased resistance to virulent Pst DC3000 and H.
arabidopsidis and constitutive expression of SA-dependent defence
genes such as PR-1, PR-5 and WRKY53 (as well as the JA/Etdependent PDF1.2) in the absence of pathogen challenge [24,25].
As reported for other cpr-type mutants, SA accumulation is
essential for the increased resistance to virulent biotrophic
pathogens displayed by cir1, which appears to be mediated by
both NPR1-dependent and independent signalling pathways, since
cir1 npr1 double mutants displayed only partial suppression of
cir1-mediated resistance [24]. Although cir1 displays increased
PDF1.2 expression, it does not display increased resistance to the
necrotrophic fungal pathogen Botrytis cinerea [23]. The CIR1
gene maps to the lower arm of chromosome IV and complementation tests have revealed that it is not allelic to previously reported
cpr mutations in this region including cpr1 [24].
Epistasis analyses have revealed that EDS1 and PAD4 are
required for constitutive PR expression and enhanced disease
resistance in several cpr mutants including cpr1 and cpr6 [21,26].
Given these results and the pivotal role of the EDS1-PAD4
regulatory node in SA-mediated defence against biotrophic
pathogens we investigated whether cir1-mediated resistance to
Pst DC3000 and H. arabidopsidis also requires EDS1 and/or
PAD4, and whether CIR1 might in turn regulate EDS1
expression. Our data indicate that CIR1 is a negative regulator
of PTI and of Toll–interleukin-1 receptor–nucleotide binding–
leucine-rich repeat (TIR-NB-LRR) R protein mediated ETI
against biotrophic and hemi-biotrophic pathogens [9–11]. Recent
evidence suggests that EDS1 may also play a role in ETI mediated
via coiled coil-NB-LRR R proteins, as EDS1 and SA accumulation have been shown to function redundantly in RPS2 and RPP8mediated resistance against avirulent pathogens [12]. Both EDS1
and PAD4 are required for SA accumulation in response to
Pseudomonas syringe pv. tomato (Pst) DC3000 or Pst DC3000
avrRps4 [13], and EDS1 and PAD4 gene expression is SAinducible, suggesting the existing of a positive feedback loop
[11,13].
Protein-protein interaction studies have identified the presence
of EDS1 homodimers, as well as EDS1-PAD4, EDS1-SAG101
and EDS1-PAD4-SAG101 protein complexes in plant cells [13–
16]. The formation of the EDS1-PAD4 complex is required for
PTI against virulent pathogens, full accumulation of SA and the
establishment of SAR, but not for TIR-NB-LRR mediated ETI
[15]. While EDS1 homodimers are present predominantly in the
cytoplasm, the EDS1-PAD4 complex is found in the cytoplasm
and nucleus, and it has been suggested that nuclear EDS1 acts as a
transcriptional regulator [14,15]. Enhanced export of EDS1 from
the nucleus was found to increase susceptibility to both virulent
and avirulent Pst DC3000, as well as Hyaloperonospora arabidopsidis Emwa1 [17], but co-ordination of cytoplasmic and
nuclear EDS1 levels may also be important in the plant immune
response [17]. In line with its central role in innate immunity in
Arabidopsis, EDS1 is targeted by the Pst effectors AvrRps4 and
HopA1, and in accordance with the guard hypothesis of Van der
Biezen and Jones [18], EDS1 is found in association with the
cognate TIR-NB-LRR R proteins RPS4 and RPS6 [19].
EDS1 and PAD4 were identified in Arabidopsis by screening for
altered susceptibility to pathogen challenge, and mutant screens
have been widely used to dissect the defence signalling network.
One class of gain-of-resistance mutants that display SAR-like
Figure 1. EDS1 and PAD4 are required for cir1-mediated resistance to Pst DC3000. Four-week old plants grown at 22uC were pressure
inoculated with Pst DC3000 (106 cfu mL21) and bacterial titres determined at 48 hpi. Data shown are mean values 6 SEM (n = 3) from one experiment
representative of three independent experiments. Mean bacterial titres (cfu cm22) with different letters are significantly different (p,0.05).
doi:10.1371/journal.pone.0109853.g001
PLOS ONE | www.plosone.org
2
October 2014 | Volume 9 | Issue 10 | e109853
CIR1-Mediated Immunity in Arabidopsis Requires EDS1 and PAD4
Figure 2. EDS1 and PAD4 are required for cir1-mediated resistance to H. arabidopsidis Noco2. Four-week old plants grown at 22uC were
infected with H. arabidopsidis (104 conidiospores mL21) and condiospore load determined at 7 dpi. ANOVA revealed a significant effect of host
genotype (p,0.001) on conidiospore load. Mean conidiospore counts (spores g21 fresh weight) with different letters are significantly different (p,
0.05). Data shown are mean values 6 SEM (n = 4) from one experiment representative of three independent experiments.
doi:10.1371/journal.pone.0109853.g002
of innate immunity that lies upstream of EDS1 and PAD4 in the
defence signalling network, and suggest that CIR1 may be
involved in the post-transcriptional regulation of EDS1. In
addition, we show that the defence and growth phenotypes of
the cir1 mutant are modulated by environmental temperature.
Results
cir1-mediated resistance to Pst DC3000 and H.
arabidopsidis requires EDS1 and PAD4
The increased resistance to Pst DC3000 and H. arabidopsidis
Noco2 displayed by the cir1 mutant has previously been shown to
be SA-dependent [24]. EDS1 and PAD4 are two key players in
Figure 3. EDS1 and PAD4 are required for cir1-mediated constitutive defence gene expression. Relative expression values for At2g14160
(PR-1) and At2g31880 (suppressor of BIR1) were determined in four-week-old plants grown at 22uC using qPCR, with normalisation to Actin2
expression. Col-0 + Pst plants were inoculated with Pst DC3000 (106 cfu mL21) and tissue harvested after 24 h. Values shown are the means of two
independent biological repeats + SD.
doi:10.1371/journal.pone.0109853.g003
PLOS ONE | www.plosone.org
3
October 2014 | Volume 9 | Issue 10 | e109853
CIR1-Mediated Immunity in Arabidopsis Requires EDS1 and PAD4
(Figure 1). Thus, both EDS1 and PAD4 are required for cir1mediated resistance to Pst DC3000, and are epistatic to CIR1.
We also examined the resistance profile of the double mutants
to H. arabidopsidis Noco2. This pathogen is virulent on the
Arabidopsis Col-0 ecotype, but avirulent on Ler due to the
presence of the RPP5 resistance gene [27]. EDS1 is required for
RPP5-mediated resistance to H. arabidopsidis [9], as reflected in
the significantly higher conidiospore production and mycelial
growth observed in eds1-2 compared to Ler (Figure 2 and S1).
Conidiospore production in the five independently derived cir1
eds1 lines was not significantly different from that observed in
eds1-2, while that in cir1 was significantly lower (Figure 2 and S1).
Again, similar results were observed with the pad4 mutants, with
no statistically significant difference in conidiospore production
between pad4-1 and cir1 pad4. However, disease symptoms were
less severe in pad4-1 in comparison to eds1-2 as previously
reported [15]. Together these data suggest that EDS1 and PAD4
are also required for cir1-mediated resistance to H. arabidopsidis,
and again function downstream of CIR1. We observed that cir1 in
the Ler (cir1 Ler) background displayed the reduced susceptibility
to H. arabidopsidis observed in cir1 in the Col-0 backround,
rather than the total resistance displayed by wild-type Ler plants
(Figure 2). Intermediate susceptibility to this pathogen has
previously been reported in Col-06Ler crosses [27].
EDS1 and PAD4 are required for cir1-modulated defence
gene expression
As both EDS1 and PAD4 are required for cir1-mediated
resistance against virulent biotrophic pathogens, we examined the
expression of two downstream defence genes that are SA
responsive and require EDS1 and PAD4 for up-regulation in
response to Pst DC3000 infection [28,29]. Quantitative PCR
analysis confirmed that At2g14160 (PR-1) and At2g31880
(suppressor of BAK1-interacting receptor-like kinase 1, SOBIR1)
were up-regulated in Col-0 plants 24 hpi with Pst DC3000, and
that mRNA levels were elevated in uninfected cir1 plants
compared to uninfected Col-0 plants (Figure 3). However, the
cir1 eds1 and cir1 pad4 double mutants again phenocopied the
single eds1-2 or pad4-1 mutants rather than cir1, as mRNA levels
of both genes were not elevated in these plants (Figure 3). These
data indicate that EDS1 and PAD4 are required for elevated
constitutive expression of these defence genes in cir1, and support
the hypothesis that CIR1 is upstream of the EDS1-PAD4
regulatory node.
Figure 4. EDS1 protein but not mRNA levels are constitutively
higher in the cir1 mutant. (A) Total protein from 4-week-old plants
grown at 22uC was separated by SDS–PAGE, transferred to nitrocellulose membrane and probed with an EDS1 antibody. Equal loading of
the gel was verified by Ponceau staining of the membrane after protein
transfer. This experiment was repeated twice with the same results. (B)
Relative EDS1 expression in 4-week-old cir1 and PR-1:LUC plants was
determined using qPCR, with normalization to Actin2 expression levels.
Each value is the mean of three independent biological repeats 6 SEM.
This experiment was repeated three times with the same results.
doi:10.1371/journal.pone.0109853.g004
defence against Pst DC3000 and H. arabidopsidis and are essential
for SA accumulation in response to infection by these pathogens
[13]. To determine whether cir1-mediated resistance to these
pathogens is also dependent on EDS1 and PAD4, we generated
cir1 eds1 and cir1 pad4 double mutants and examined their
disease susceptibility profiles.
As eds1 is in the Ler background, while cir1 is in the Col-0
background, we analysed five independently generated cir1 eds1
double mutants to control for any effects of a mixed Col-0/Ler
background on resistance to Pst DC3000. Bacterial titres in these
five lines at 48 h post-infection (hpi) were not significantly different
from those observed in the eds1-2 mutant, while those observed in
cir1 were significantly lower (Figure 1). There was no significant
difference in bacterial titres between the cir1 mutant in the Col-0
background (cir1) and cir1 plants generated by crossing the single
mutant to wild-type Ler plants (cir1 Ler), indicating that a mixed
Col-0/Ler genetic background has no effect on cir1-mediated
resistance to Pst DC3000 (Figure 1). Similar results were observed
for PAD4; cir1 displayed significantly lower bacterial titres 48 hpi
than pad4-1, while no statistically significant difference in bacterial
titres was observed between pad4-1 and cir1 pad4 plants
PLOS ONE | www.plosone.org
EDS1 expression is constitutively higher in the cir1
mutant
Given that CIR1 functions upstream of EDS1 and PAD4, we
next examined whether CIR1 might regulate EDS1 expression in
Arabidopsis. Western blot analysis revealed that EDS1 proteins
levels were constitutively higher in uninfected cir1 plants in
comparison to PR-1:LUC control plants (the genetic background
for the cir1 mutant), and also in cir1 pad4 plants versus the pad4-1
single mutant (Figure 4A). However, analysis of EDS1 steady-state
transcript levels using qPCR revealed no statistically significant
difference between cir1 and PR-1:LUC control plants (Figure 4B),
suggesting that CIR1 may exert its regulatory effect on EDS1 via a
post-transcriptional mechanism.
The cir1 growth and disease resistance phenotypes are
temperature-sensitive
A number of gain-of-resistance mutants display a stunted
growth phenotype, which is thought to result from the fitness cost
4
October 2014 | Volume 9 | Issue 10 | e109853
CIR1-Mediated Immunity in Arabidopsis Requires EDS1 and PAD4
Figure 5. The cir1 mutant displays a temperature-sensitive growth phenotype. Representative cir1 and PR-1:LUC plants grown for four
weeks under a 16 h light/8 h dark cycle at 18, 22 or 25uC are shown. Scale bar indicates a distance of 10 mm.
doi:10.1371/journal.pone.0109853.g005
while at 25uC no obvious difference in size was evident (Figure 5).
ANOVA of Pst DC3000 titres 48 hpi revealed significant (p,
0.001) effects of both temperature and genotype on resistance to
Pst DC3000, and a significant interaction term (genotype*temperature, p = 0.015), indicating that the effect of genotype on
resistance to Pst DC3000 is modulated by environmental
temperature. While bacterial titres were on average 8.1-fold (6
2.3) lower in cir1 plants compared to PR-1:LUC plants when
grown at 22uC, at 18uC bacterial titres in the cir1 mutant were on
average 30.1-fold (68.1) lower than those in control plants
(Figure 6). In contrast, when grown at 25uC there was no
of constitutive activation of immune responses [30]. In several
mutants, including cpr1, bonzai1 (bon1) and suppressor of npr1-1,
constitutive 1 (snc1), this phenotype is temperature dependent,
manifesting only at lower growth temperatures [22,31]. While cir1
does not exhibit the dwarf stature characteristic of these mutants at
22uC (our standard growth temperature for Arabidopsis), it does
display moderately reduced stature in comparison to PR-1:LUC
control plants (Figure 5). We thus examined the growth phenotype
and resistance to Pst DC3000 of the cir1 mutant when grown at
18, 22 or 25uC. We observed that cir1 plants displayed greatly
reduced stature compared to PR-1:LUC control plants at 18uC,
Figure 6. Susceptibility to Pst DC3000 is modulated by temperature in cir1. Four-week-old cir1 and PR-1:LUC plants grown at 18, 22 or 25uC
were pressure inoculated with Pst DC3000 (106 cfu mL-1) and bacterial titres determined at 48 hpi. Data shown are mean values 6 SEM (n = 8–10).
ANOVA revealed a significant effect of host genotype (p,0.001) and temperature (p,0.001) on bacterial titres at 48 hpi, A significant interaction
between these two variables (p = 0.015) indicates that they combine non-additively to influence bacterial growth. Mean bacterial titres (cfu cm22)
with different letters are significantly different (p,0.05).
doi:10.1371/journal.pone.0109853.g006
PLOS ONE | www.plosone.org
5
October 2014 | Volume 9 | Issue 10 | e109853
CIR1-Mediated Immunity in Arabidopsis Requires EDS1 and PAD4
mechanism. One possible mechanism for CIR1 action might be
via regulation of EDS1 protein accumulation or stability. Analysis
of EDS1 protein levels in the pad4-1 and cir1 pad4 mutants
(Figure 4A) offers limited support to this hypothesis; while EDS1
protein levels were reduced in the pad4-1 single mutant, in line
with previous reports that PAD4 stabilises EDS1 [14], the cir1
pad4 double mutant displayed similar EDS1 levels to cir1 plants
(Figure 4A). This suggests that the cir1 mutation may be able to
compensate for the lack of PAD4 in the stabilisation of EDS1, and
that CIR1 might serve as a negative regulator of EDS1 protein
levels in planta. Recent studies have indicated that the balance
between nuclear and cytoplasmic EDS1 pools is important in
EDS1 function in innate immunity [17]. EDS1 protein levels are
higher in both nuclei-enriched and nuclei-depleted fractions in the
snc1 mutant [17], and interaction between SNC1 and EDS1 has
been detected in both nucleus and cytoplasm [19]. However, the
sub-cellular distribution of EDS1 has not been investigated in cir1,
and so it is unclear whether EDS1 protein levels are elevated in
both sub-cellular compartments, or only in one.
Environmental conditions modulate plant-pathogen interactions, with temperature known to play an important role in
determining the strength of the host response to pathogen
challenge [30,35]. Higher temperatures within the ambient growth
temperature range of the plant have been shown to reduce the
effectiveness of both PTI and ETI [35,36]. The gain-of-resistance
mutants cpr1, bon1 and srfr1 all display temperature modulated
growth and defence phenotypes, with dwarfism and increased
resistance to biotrophic pathogens observed at 22uC but not 28uC.
Similarly, we observed that growth and resistance to Pst DC3000
infection are modulated by temperature in cir1 (Figure 5 & 6). At
18uC an obvious reduction in biomass production in cir1 was
accompanied by enhanced resistance to Pst DC3000 relative to
cir1 plants grown at 22uC. In contrast, at 25uC plant size and
resistance to Pst DC3000 were not significantly different between
cir1 and control plants. While these phenotypes are similar to
those reported for cpr1, bon1 and srfr1, the temperature at which
they occur is lower in cir1; while cpr1, bon1 and srfr1 all exhibit a
dwarf phenotype at 22uC, cir1 displays only a modest reduction in
size at this temperature.
The apparently identical temperature sensitivity of cpr1, bon1
and srfr1 may result from their convergence on the TIR-NB-LRR
protein SNC1. Transcript and/or protein levels of SNC1 are
elevated in cpr1, srfr1 and bon1 mutants, indicating that all three
proteins act as negative regulators of SNC1 [31,37,38]. CPR1 is
an F-box protein which interacts in vivo with SNC1 [37]
suggesting that it regulates SNC1 protein levels via 26S
proteasome-mediated degradation. Arabidopsis mutants expressing a constitutively active version of SNC1 (snc1-1) display
dwarfism, constitutive defence gene expression and increased
resistance to biotrophic pathogens [34]. As with cir1, cpr1, bon1
and srfr1, these phenotypes are temperature dependent, manifesting at 22uC but not at 28uC [31,36]. Analysis of the progeny
from a cross between bon1 and snc1-11 (a null allele) has revealed
that SNC1 is essential for the dwarfism, constitutive PR gene
expression and enhanced resistance to biotrophic pathogens
displayed by bon1 at 22uC [31]. Similarly, dwarfism and
constitutive PR-1 gene expression are abolished in the srfr1
snc1-11 and cpr1 snc1-11 double mutants [22,38], suggesting that
the auto-immune phenotypes of cpr1, bon1 and srfr1 at 22uC
result largely from de-repression of SNC1.
SNC1 therefore appears to act as a temperature sensor in
Arabidopsis to modulate host immunity in response to changes in
the environment. Further evidence for this role comes from the
snc1-3 mutant which displays dwarfism and increased resistance to
Figure 7. SNC1 transcript levels are not elevated in cir1. Relative
SNC1 expression in 4-week-old cir1 and PR-1:LUC plants grown at 18 or
22uC was determined using qPCR, with normalization to Actin2
expression levels. Each value is the mean of three independent
biological repeats 6 SEM.
doi:10.1371/journal.pone.0109853.g007
significant difference in bacterial titres 48 hpi (Figure 6), suggesting
that enhanced resistance to Pst DC3000 was abolished in the cir1
mutant at this temperature. The auto-immune phenotypes
displayed by several cpr-type mutants including cpr1 and bon1
when grown at 22uC have been linked to increased expression of
the TIR-NB-LRR protein SNC1, with increased SNC1 mRNA
levels reported in cpr1 and bon1 [31,37]. However, qPCR analysis
revealed that SNC1 transcript levels were not significantly higher
in cir1 versus PR-1:LUC plants when grown at either 18 or 22uC
(Figure 7).
Discussion
The EDS1-PAD4 regulatory node plays a critical role in both
PTI and ETI against biotrophic pathogens in Arabidopsis. Here
we investigated whether the enhanced resistance to Pst DC3000
and H. arabidopsidis and constitutive expression of SA-dependent
defence genes displayed by the cir1 mutant requires EDS1 and
PAD4 by generating double mutants. We observed that both cir1
eds1 and cir1 pad4 plants displayed the enhanced susceptibility to
Pst DC3000 and H. arabidopsidis that is characteristic of the
single eds1-2 and pad4-1 null mutants (Figures 1 & 2). Similarly,
analysis of steady state levels of two SA-dependent defence genes
revealed that their elevated expression in uninfected cir1 plants
requires both EDS1 and PAD4 (Figure 3). These results are in line
with those previously observed for a number of other gain-ofresistance mutants including cpr1, cpr6, bon1, snc1 and suppressor
of rps4-RLD 1 (srfr1) [21,22,32–34], and indicate that the SARlike constitutive disease resistance displayed by cir1 also operates
via the EDS1-PAD4 regulatory node.
Given that CIR1 is epistatic to EDS1, we investigated whether
CIR1 might regulate EDS1 expression in Arabidopsis. Western
blot analysis of the cir1 mutant revealed that EDS1 protein levels
in uninfected plants were higher than in PR-1:LUC control plants
(Fig. 4A). As cir1 displays constitutively elevated expression of SAdependent genes, and EDS1 gene expression is SA-inducible [11],
elevated EDS1 protein levels in cir1 may simply be a consequence
of upregulated EDS1 transcription in this mutant. However,
qPCR analysis revealed no significant difference in steady state
levels of the EDS1 transcript between the cir1 and PR-1:LUC
plants (Figure 4B), suggesting that CIR1 may instead function as a
negative regulator of EDS1 via a post-transcriptional regulatory
PLOS ONE | www.plosone.org
6
October 2014 | Volume 9 | Issue 10 | e109853
CIR1-Mediated Immunity in Arabidopsis Requires EDS1 and PAD4
Pst DC3000 at both 22uC and 28uC [36]. It has been suggested
that a threshold concentration of SNC1 must be reached in the
nucleus to trigger immunity, supported by data showing that in
wild-type plants SNC1 nuclear content decreases with increasing
temperature, but not in the snc1-3 mutant [36]. Whether the
growth and constitutive defence phenotypes of cir1 also require
SNC1 is currently unknown, although we did not observe a
statistically significant increase in SNC1 transcript levels in cir1
versus PR-1:LUC plants at either 18 or 22uC (Figure 7). Analysis
of the cir1 snc1-11 double mutants we are currently generating
will address the role of SNC1 in cir1. We are also carrying out
genetic mapping to identify the CIR1 gene. First-pass mapping
experiments on the F2 progeny of a cross between cir1 and Ler
plants indicated that cir1 mapped approximately 9.4 cM below
nga111 on the lower arm of chromosome 4 [24]. Subsequent
linkage analysis to markers within this region has localised CIR1 to
a 46 kb region of chromosome IV, and we are currently analysing
candidate genes within this region. Identification of the CIR1 gene
will shed light on the exact biochemical roles played by this
negative regulator of EDS1 and PAD4-mediated immunity in
Arabidopsis.
from the expected 13:3 ratio of low:high PR-1:LUC activity
suggesting that the eds1 and pad4 mutations were affecting PR1::LUC reporter activity. A two-step process was therefore
employed to isolate the double mutants. Homozygous cir1 plants
were identified in the F2 generation by screening for high PR1:LUC activity, comparable to that of the single cir1 mutant. As
expected, genotyping revealed that all were heterozygous for either
the eds1-2 or pad4-1 mutant allele. The F3 progeny from these
plants were then screened by PCR genotyping or RFLP analysis to
identify individuals homozygous for the eds1 or pad4 mutation.
The PCR primers used for genotyping the EDS1 locus were 59GTGGAAACCAAATTTGACATTAG-39 and 59-ACACAAGGGTGATGCGAGACA-39 which generated PCR products of
750 bp (EDS1) or 600 bp (eds1-2). For PAD4 genotyping, the
primers used were 59-GCGATGCATCAGAAGAG-39 and 59TTAGCCCAAAAGCAAGTATC-39 which generated a 391 bp
PCR product. The PAD4 amplicon is cleaved by BsmFI to give
products of 281 and 110 bp, while the pad4-1 amplicon is not. As
eds1-2 is in the Ler background, cir1 was also crossed with Ler to
determine whether the mixed Col-0/Ler genetic background
affected the penetrance of the cir1 mutation.
Materials and Methods
Quantitative PCR analysis
Plant growth conditions
Total RNA was extracted using Trizol (Invitrogen), treated with
DNase and cDNA synthesised from 1 mg of RNA using
Superscript III reverse transcriptase (Invitrogen). Quantitative
PCR was performed using a RotorGene RG3000A instrument
(Corbett Research, Australia). Reactions consisted of 1 mL
template cDNA, 5 mL Kapa SYBR FAST Universal 26qPCR
Master Mix (Kapa Biosystems, South Africa), and 200–900 nM of
each primer in a final volume of 10 mL. Amplification conditions
included an initial step at 95uC for 3 min, followed by 40 cycles of
95uC for 3 s, primer annealing at 60 or 65uC for 20 s and
elongation at 72uC for 1 s. Melt curve analysis confirmed that the
individual amplified products corresponded to a single, genespecific cDNA fragment. The relative expression level of each gene
of interest was calculated with the RotorGene 6000 series software
v1.7 using the two standard curve method, with normalisation to
the reference gene Actin-2 (At3g18780). Details of the primers
used and specific qPCR reaction conditions can be found in Table
S1.
Arabidopsis seeds were stratified for 48 h at 4uC in the dark
prior to sowing on either a 1:1 mix of peat (Jiffy Products, Norway)
and vermiculite or on half-strength MS agar plates. Plants were
grown under a long-day photoperiod (16 h light, 8 h dark) at 22uC
(unless otherwise stated) and 55% relative humidity, and cool
white fluorescent light of 80–100 mmol m22s21.
Pathogen assays
All pathogen assays were carried out on four-week old
Arabidopsis plants. Pseudomonas syringae pv. tomato DC3000
infections were carried out as previously described [39].
Hyaloperonospora arabidopsidis Noco2 infections were carried
out as described by Parker et al. [27], and the extent of plant cell
necrosis and development of H. arabidopsidis mycelium was
examined microscopically 7 dpi by lactophenol trypan blue
staining [10].
Luciferase assays
Western Blot analyses
Total protein was extracted by homogenizing leaf tissue from
four-week-old soil grown plants in 1 mL extraction buffer (100
mM sodium phosphate buffer pH 7.2, 5 mM DTT). The samples
were centrifuged for 5 min at 12 0006g to pellet cell debris, and
100 mL of the resulting supernatant added to 100 mL of assay
buffer (60 mM Tris-HCl pH 8.0, 20 mM MgCl2, 20 mM DTT, 2
mM EDTA, 2 mM ATP). Luciferase activity was measured for 20
s following injection with 100 mL of luciferin buffer (60 mM TrisHCl pH 8.0, 20 mM MgCl2, 20 mM DTT, 2 mM EDTA, 1 mM
luciferin) in a Luminoskan TL-Plus luminometer (Labsystems,
Finland). Luciferase activity was normalised to total protein
concentration as determined by Bradford protein assay.
Total protein was isolated from leaf tissue as described by Ingle
et al. [40]. Forty mg of total protein was separated on 12% (w/v)
SDS PAGE gels, transferred to nitrocellulose membrane and
blocked for 2 h at RT in 16TBS-T buffer containing 2% (w/v)
skim milk powder. Primary EDS1 antibody [13] was diluted 1:400
in 16TBS-T buffer with 2% (w/v) milk powder, and blots
incubated o/n at 4uC. Incubation with primary antibody was
followed by 3610 min washes in 16TBST, and incubation with
secondary antibody (Rabbit IgG HRP, 1:5000 dilution) for 1 h at
RT, prior to band detection by chemiluminescence.
Statistical analyses
Generation of cir1 eds1 and cir1 pad4 double mutants
Statistical analyses of all data were carried out using Statistica
(version 9). Pst DC3000 titre data were log-transformed prior to
ANOVA to ensure homogeneity of variance and normality of
error. Fisher’s LSD post-hoc analysis was used to identify
significantly different mean values within an experiment. The
raw data obtained from pathogen assays and qPCR experiments
that were used in the statistical analyses (and in the generation of
Figures 1, 2, 3, 4B, 6 and 7) are provided in Table S2.
Homozygous cir1 plants were crossed to either eds1-2 (Ler
background) or pad4-1 (Col-0 background) mutants, and the
resulting F1 progeny allowed to self-fertilise. While the marker
used to screen for homozygosity for the cir1 mutation is elevated
PR-1:LUC activity, both EDS1 and PAD4 are known to be
required for PR-1 expression [13]. Unsurprisingly then, segregation analyses of the F2 progeny revealed a significant deviation
PLOS ONE | www.plosone.org
7
October 2014 | Volume 9 | Issue 10 | e109853
CIR1-Mediated Immunity in Arabidopsis Requires EDS1 and PAD4
Supporting Information
Table S2 Data used to generate Figures 1, 2, 3, 4B, 6
Figure S1 Trypan blue-stained leaf tissue of four-week-
and 7.
(XLSX)
old plants six days post-infection with Hyaloperonospora
arabidopsidis Noco2.
(PDF)
Author Contributions
Conceived and designed the experiments: RI KD SM JP. Performed the
experiments: MC TM NA AD DG. Analyzed the data: MC TM NA RI
KD. Contributed reagents/materials/analysis tools: JP. Contributed to the
writing of the manuscript: KD RI.
Table S1 Primers used in quantitative PCR experi-
ments.
(PDF)
References
22. Gou M, Su N, Zheng J, Huai J, Wu G, et al. (2009) An F-box gene, CPR30,
functions as a negative regulator of the defense response in Arabidopsis. Plant J
60: 757–770.
23. Murray SL, Adams N, Kliebenstein DJ, Loake GJ, Denby KJ (2005) A
constitutive PR-1::luciferase expression screen identifies Arabidopsis mutants
with differential disease resistance to both biotrophic and necrotrophic
pathogens. Mol Plant Pathol 6: 31–41.
24. Murray SL, Thomson C, Chini A, Read ND, Loake GJ (2002) Characterization
of a novel, defense-related Arabidopsis mutant, cir1, isolated by luciferase
imaging. Mol Plant-Microbe Interact 15: 557–566.
25. Murray SL, Ingle RA, Petersen LN, Denby KJ (2007) Basal resistance against
Pseudomonas syringae in Arabidopsis involves WRKY53 and a protein with
homology to a nematode resistance protein. Mol Plant-Microbe Interact 20:
1431–1438.
26. Jirage D, Zhou N, Cooper B, Clarke JD, Dong X, et al. (2001) Constitutive
salicylic acid-dependent signaling in cpr1 and cpr6 mutants requires PAD4.
Plant J 26: 395–407.
27. Parker JE, Szabò V, Staskawicz BJ, Lister C, Dean C, et al. (1993) Phenotypic
characterization and molecular mapping of the Arabidopsis thaliana locus
RPP5, determining disease resistance to Peronospora parasitica. Plant J 4: 821–
831.
28. Bartsch M, Gobbato E, Bednarek P, Debey S, Schultze JL, et al. (2006) Salicylic
acid-independent ENHANCED DISEASE SUSCEPTIBILITY1 signaling in
Arabidopsis immunity and cell death is regulated by the monooxygenase FMO1
and the nudix hydrolase NUDT7. Plant Cell 18: 1038–1051.
29. Glazebrook J, Chen W, Estes B, Chang HS, Nawrath C, et al. (2003) Topology
of the network integrating salicylate and jasmonate signal transduction derived
from global expression phenotyping. Plant J 34: 217–228.
30. Alcázar R, Parker JE (2011) The impact of temperature on balancing immune
responsiveness and growth in Arabidopsis. Trends Plant Sci 16: 666–675.
31. Yang S, Hua J (2004) A haplotype-specific resistance gene regulated by
BONZAI1 mediates temperature-dependent growth control in Arabidopsis.
Plant Cell 16: 1060–1071.
32. Kwon SI, Kim SH, Bhattacharjee S, Noh J-J, Gassmann W (2009) SRFR1, a
suppressor of effector-triggered immunity, encodes a conserved tetratricopeptide
repeat protein with similarity to transcriptional repressors. Plant J 57: 109–119.
33. Li X, Clarke JD, Zhang Y, Dong X (2001) Activation of an EDS1-mediated Rgene pathway in the snc1 mutant leads to constitutive, NPR1-independent
pathogen resistance. Mol Plant-Microbe Interact 14: 1131–1139.
34. Zhang Y, Goritschnig S, Dong X, Li X (2003) A gain-of-function mutation in a
plant disease resistance gene leads to constitutive activation of downstream signal
transduction pathways in suppressor of npr1-1, constitutive 1. Plant Cell 15:
2636–2646.
35. Wang Y, Bao Z, Zhu Y, Hua J (2009) Analysis of temperature modulation of
plant defense against biotrophic microbes. Mol Plant-Microbe Interact 22: 498–
506.
36. Zhu Y, Qian W, Hua J (2010) Temperature modulates plant defense responses
through NB-LRR proteins. PLoS Pathog 6: e1000844.
37. Cheng YT, Li Y, Huang S, Huang Y, Dong X, et al. (2011) Stability of plant
immune-receptor resistance proteins is controlled by SKP1-CULLIN1-F-box
(SCF)-mediated protein degradation. Proc Natl Acad Sci 108: 14694–14699.
38. Kim SH, Gao F, Bhattacharjee S, Adiasor JA, Nam JC, et al. (2010) The
Arabidopsis resistance-like gene SNC1 is activated by mutations in SRFR1 and
contributes to resistance to the bacterial effector AvrRps4. PLoS Pathog 6:
e1001172.
39. Bhardwaj V, Meier S, Petersen LN, Ingle RA, Roden LC (2011) Defence
responses of Arabidopsis thaliana to infection by Pseudomonas syringae are
regulated by the circadian clock. PLoS ONE 6: e26968.
40. Ingle RA, Smith JAC, Sweetlove LJ (2005) Responses to nickel in the proteome
of the hyperaccumulator plant Alyssum lesbiacum. Biometals 18: 627–641.
1. Jones JD, Dangl JL (2006) The plant immune system. Nature 444: 323–329.
2. Schwessinger B, Ronald PC (2012) Plant innate immunity: perception of
conserved microbial signatures. Ann Rev Plant Biol 63: 451–482.
3. Schwessinger B, Zipfel C (2008) News from the frontline: recent insights into
PAMP-triggered immunity in plants. Curr Opin Plant Biol 11: 389–395.
4. Spoel SH, Dong X (2012) How do plants achieve immunity? Defence without
specialized immune cells. Nature Rev Immunol 12: 89–100.
5. Fu ZQ, Dong X (2013) Systemic acquired resistance: turning local infection into
global defense. Ann Rev Plant Biol 64: 839–863.
6. Robert-Seilaniantz A, Grant M, Jones JD (2011) Hormone crosstalk in plant
disease and defense: more than just jasmonate-salicylate antagonism. Ann Rev
Phytopathol 49: 317–343.
7. Windram O, Madhou P, McHattie S, Hill C, Hickman R, et al. (2012)
Arabidopsis defense against Botrytis cinerea: chronology and regulation
deciphered by high-resolution temporal transcriptomic analysis. Plant Cell 24:
3530–3557.
8. Glazebrook J, Rogers EE, Ausubel FM (1996) Isolation of Arabidopsis mutants
with enhanced disease susceptibility by direct screening. Genetics 143: 973–982.
9. Parker JE, Holub EB, Frost LN, Falk A, Gunn ND, et al. (1996)
Characterization of eds1, a mutation in Arabidopsis suppressing resistance to
Peronospora parasitica specified by several different RPP genes. Plant Cell 8:
2033–2046.
10. Aarts N, Metz M, Holub E, Staskawicz BJ, Daniels MJ, et al. (1998) Different
requirements for EDS1 and NDR1 by disease resistance genes define at least two
R gene-mediated signaling pathways in Arabidopsis. Proc Natl Acad Sci 95:
10306–10311.
11. Wiermer M, Feys BJ, Parker JE (2005) Plant immunity: the EDS1 regulatory
node. Curr Opin Plant Biol 8: 383–389.
12. Venugopal SC, Jeong R-D, Mandal MK, Zhu S, Chandra-Shekara A, et al.
(2009) Enhanced disease susceptibility 1 and salicylic acid act redundantly to
regulate resistance gene-mediated signaling. PLoS Genet 5: e1000545.
13. Feys BJ, Moisan LJ, Newman M-A, Parker JE (2001) Direct interaction between
the Arabidopsis disease resistance signaling proteins, EDS1 and PAD4. EMBO J
20: 5400–5411.
14. Feys BJ, Wiermer M, Bhat RA, Moisan LJ, Medina-Escobar N, et al. (2005)
Arabidopsis SENESCENCE-ASSOCIATED GENE101 stabilizes and signals
within an ENHANCED DISEASE SUSCEPTIBILITY1 complex in plant
innate immunity. Plant Cell 17: 2601–2613.
15. Rietz S, Stamm A, Malonek S, Wagner S, Becker D, et al. (2011) Different roles
of Enhanced Disease Susceptibility1 (EDS1) bound to and dissociated from
Phytoalexin Deficient4 (PAD4) in Arabidopsis immunity. New Phytol 191: 107–
119.
16. Zhu S, Jeong R, Venugopal S, Lapchyk L, Navarre D, et al. (2011) SAG101
forms a ternary Complex with EDS1 and PAD4 and is required for resistance
signaling against Turnip Crinkle Virus. PloS Pathog 7: e1002318.
17. Garcı́a AV, Blanvillain-Baufumé S, Huibers RP, Wiermer M, Li G, et al. (2010)
Balanced nuclear and cytoplasmic activities of EDS1 are required for a complete
plant innate immune response. PLoS Pathog 6: e1000970.
18. Van Der Biezen EA, Jones JD (1998) Plant disease-resistance proteins and the
gene-for-gene concept. Trends Biochem Sci 23: 454–456.
19. Bhattacharjee S, Halane MK, Kim SH, Gassmann W (2011) Pathogen effectors
target Arabidopsis EDS1 and alter its interactions with immune regulators.
Science 334: 1405–1408.
20. Bowling SA, Guo A, Cao H, Gordon AS, Klessig DF, et al. (1994) A mutation in
Arabidopsis that leads to constitutive expression of systemic acquired resistance.
Plant Cell 6: 1845–1857.
21. Clarke JD, Aarts N, Feys BJ, Dong X, Parker JE (2001) Constitutive disease
resistance requires EDS1 in the Arabidopsis mutants cpr1 and cpr6 and is
partially EDS1-dependent in cpr5. Plant J 26: 409–420.
PLOS ONE | www.plosone.org
8
October 2014 | Volume 9 | Issue 10 | e109853